Generation of Mouse Small Intestinal Epithelial Cell Lines That Allow the Analysis of Specific Innate Immune Functions

Cell lines derived from the small intestine that reflect authentic properties of the originating intestinal epithelium are of high value for studies on mucosal immunology and host microbial homeostasis. A novel immortalization procedure was applied to generate continuously proliferating cell lines from murine E19 embryonic small intestinal tissue. The obtained cell lines form a tight and polarized epithelial cell layer, display characteristic tight junction, microvilli and surface protein expression and generate increasing transepithelial electrical resistance during in vitro culture. Significant up-regulation of Cxcl2 and Cxcl5 chemokine expression upon exposure to defined microbial innate immune stimuli and endogenous cytokines is observed. Cell lines were also generated from a transgenic interferon reporter (Mx2-Luciferase) mouse, allowing reporter technology-based quantification of the cellular response to type I and III interferon. Thus, the newly created cell lines mimic properties of the natural epithelium and can be used for diverse studies including testing of the absorption of drug candidates. The reproducibility of the method to create such cell lines from wild type and transgenic mice provides a new tool to study molecular and cellular processes of the epithelial barrier.     

Energization and activation of inorganic carbon uptake by light in cyanobacteria

The requirement of the inorganic carbon (C_i) transport system for light in cyanobacteria was investigated in Anabaena variabilis by the filtering centrifugation technique and in a mutant (E₁) isolated from Anacystis nidulans using a gas exchange system. C_i transport capability increased with time of preillumination and decreased following darkening. Full activity could not be obtained by operating either photosystem II (PSII) or photosystem I alone. 3(3,4 Dichlorophenyl)-1,1 dimethylurea strongly inhibited C_i uptake. Very low activity of PSII was sufficient to activate C_i uptake. However, in the presence of dithiothreitol PSII activity was not required. We conclude that light may be required to activate as well as to energize C_i uptake in cyanobacteria.     

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbillus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially reinactivated, suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.Deceased on 2 Jan. 1987     

Cloning of a human S-phase cell cycle gene: use of transient expression for screening.

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.     

Platelet-neutrophil-smooth muscle cell interactions: lipoxygenase-derived mono- and dihydroxy acids activate cholesteryl ester hydrolysis by the cyclic AMP dependent protein kinase cascade

Fluid-phase interactions between hematologic cells and those of the vessel wall were studied in order to define a role for lipoxygenase products as cell signals in the control of vascular cholesterol metabolism. A functional parameter for hydroxy acids in this system has not been previously demonstrated. We report herein for the first time a biochemical effect of lipoxygenase-derived eicosanoids in the modulation of cholesterol metabolism in smooth muscle cells. Products of platelet-neutrophil interactions served as cell signals in vitro to modulate cholesterol metabolism. We demonstrate that 12-HETE, 12,20-DiHETE, and 12-HETE-1,20-dioic acid activate both lysosomal and cytoplasmic cholesteryl ester (CE) hydrolytic activities, although no effect was observed on CE synthetic (ACAT) activity. The platelet lipoxygenase product, 12-HETE, was the most effective stimulator of CE hydrolysis in the smooth muscle cell, and its conversion to 12,20-DiHETE and the dioic acid derivative by the neutrophils was not necessary for the activation of CE hydrolase. A 2-fold enhancement on CE hydrolysis occurred and was independent of any "cross-activation" by hydroxy acids on production of cyclooxygenase or other lipoxygenase products. The activation of cytoplasmic CE hydrolysis had a lesser cofactor dependence on bile salts in the presence of 12-HETE. This suggested a reduced requirement for surface-active agents in an enzyme-substrate interaction where enzymes are hydrolyzing insoluble lipid substrates. Moreover, 12-HETE induced an additive effect with several lipolytic hormones in the activation of CE catabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock

Attempts to transform wild type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were not successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type and DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.